Effector T lymphocytes function by secretion of mediators which influence other cells. Secretion occurs via two pathways the "constitutive" pathway, in which newly synthesized proteins pass through the Golgi and are immediately released by exocytosis of small vesicles; and the "regulated" pathway in which mediators are stored in larger granules until TcR engagement signals their exocytosis. In lymphocytes the regulated pathway has been exclusively associated with cytotoxicity and the secretion of perforin and granzymes (granule exocytosis pathway). We investigated the T cell pathway used for chemokine secretion, particularly the major T cell product RANTES. Purified populations of both CD4+ and CD8+ human T cell blasts were found to secrete RANTES within two hours after TcR stimulation by plate-bound anti-CD3. The regulated pathway was implicated by the lack of inhibition by cycloheximide (which blocks protein synthesis) and Brefeldin A (which blocks Golgi export). At times greater than 3 hours after TcR triggering these drugs block secretion, indicating a switch to the constitutive pathway. Flow cytometry of permeabilized cells shows that RANTES is present in both CD8+ and CD4+ T cell blasts and that cytoplasmic RANTES is lost rapidly after TcR crosslinking (~ 6x faster than granule markers). By confocal microscopy intracellular RANTES appears as vesicles that do not colocalize with proteins such as perforin and granzymes in the lysosomal granules. This lack of colocalization was confirmed by the higher resolution technique of deconvolution microscopy, both visually and by statistical analysis of digital pixel intensities. Immunogold staining of ultrathin EM sections confirmed that RANTES is present in vesicles that are morphologically distinct from the granules. Thus we have described a novel regulated secretory pathway in T lymphocytes, capable of rapid delivery of preformed chemokines after antigen recognition.